Actin Sliding Velocities are Influenced by the Driving Forces of Actin-Myosin Binding

Unloaded shortening speeds, V, of muscle are thought to be limited by actin-bound myosin heads that resist shortening, or V = a center dot A d center dot tau (on) (-1) where tau (on) (-1) is the rate at which myosin detaches from actin and d is myosin's step size. The a-term describes the efficiency of force transmission between myosin heads, and has been shown to become less than one at low myosin densities in a motility assay. Molecules such as inorganic phosphate (P (i) ), and blebbistatin inhibit both V and actin-myosin strong binding kinetics suggesting a link between V and attachment kinetics. To determine whether these small molecules slow V by increasing resistance to actin sliding or by decreasing the efficiency of force transmission, a, we determine how inhibition of V by P (i) and blebbistatin changes the force exerted on actin filaments during an in vitro sliding assay, measured from changes in the rate, tau (break) (-1) , at which actin filaments break. Upon addition of 30 mM P (i) to a low (30 mu M) [ATP] motility buffer V decreased from 1.8 to 1.3 mu m s(-1) and tau (break) (-1) from 0.029 to 0.018 s(-1). Upon addition of 50 mu M blebbistatin to a low [ATP] motility buffer, V decreased from 1.0 to 0.7 mu m s(-1) and tau (break) (-1) from 0.059 to 0.022 s(-1). These results imply that blebbistatin and P (i) slow V by decreasing force transmission, a, not by increasing resistive forces, implying that actin-myosin attachment kinetics influence V.